In 1985, the first X-ray structure of a membrane protein was determined. Today, more than 30 years later, many more structures have been solved. Nevertheless, studying the structure of membrane proteins remains a very challenging task. Due to their inherent conformational flexibility, having a single X-ray structure is usually only the first step towards truly understanding the function of these dynamic molecules. For this reason, additional methods are needed that can provide complementary information, especially about conformational flexibility. Pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) is such a method. It can be used to precisely measure nanometer distance distributions between intrinsic or artificially introduced spin-centers in macromolecules and thereby to probe the conformational state of the macromolecule. PELDOR can be applied in solution, in detergent, in lipid bilayers and even within cells. However, PELDOR is an advanced spectroscopy technique and requires specialised equipment and training. This chapter aims to be a starting point for crystallographers and other structural biologists who want to get a better understanding of PELDOR spectroscopy and its application. It gives an insight into the planning stages of the experiment (i.e., which spin labels are possible and where to place them), how a PELDOR experiment is conducted and how the results are interpreted. For this purpose, the substrate binding protein (SBP) from a Vibrio cholerae TRAP transporter is used as a step-by-step example. Further, the chapter gives examples of how PELDOR spectroscopy has previously been applied to overcome known limitations of X-ray crystallography in modern integrative structural biology approaches.

中文翻译：

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用 PELDOR/DEER 光谱研究膜蛋白的结构和功能——晶体学家的观点

1985 年，第一个膜蛋白的 X 射线结构被确定。30 多年后的今天，已经解决了更多的结构。尽管如此，研究膜蛋白的结构仍然是一项非常具有挑战性的任务。由于它们固有的构象灵活性，拥有单一的 X 射线结构通常只是真正了解这些动态分子功能的第一步。出于这个原因，需要额外的方法来提供补充信息，尤其是关于构象灵活性的信息。脉冲电子电子双共振光谱（PELDOR，也称DEER）就是这样一种方法。它可用于精确测量大分子中内在或人工引入的自旋中心之间的纳米距离分布，从而探测大分子的构象状态。PELDOR 可应用于溶液、洗涤剂、脂质双层甚至细胞内。然而，PELDOR 是一种先进的光谱技术，需要专门的设备和培训。本章旨在成为晶体学家和其他想要更好地了解 PELDOR 光谱及其应用的结构生物学家的起点。它可以深入了解实验的计划阶段（即，哪些自旋标签是可能的以及将它们放置在哪里）、PELDOR 实验是如何进行的以及如何解释结果。以此目的，来自霍乱弧菌 TRAP 转运蛋白的底物结合蛋白 (SBP) 用作分步示例。此外，本章还举例说明了以前如何应用 PELDOR 光谱来克服 X 射线晶体学在现代综合结构生物学方法中的已知局限性。